Fast sybr green pcr kit

Total RNA was extracted using Trizol reagent (15596026, Invitrogen). MiRcute miRNA First-strand cDNA synthesis kit (Tiangen Biotech, Beijing, China) was used for RT of miRNA, and Primer-Script^TM one step RT-PCR kit (Takara, Shiga, Japan) for RT of mRNA. The synthesized cDNA was subjected to RT-qPCR detection using Fast SYBR Green PCR kit (Applied Biosystems, Foster City, CA, United States) and 7500 Fast Real-Time PCR System (Applied Biosystems). The relative expression of genes was analyzed using the 2^–ΔΔCt method. The primer design is shown in Table 1.

Total RNA was extracted using Trizol (15,596,026, Invitrogen). Next, total RNA was reversely transcribed into cDNA according to the instructions of PrimeScript RT reagent Kit (RR047A, Takara Bio Inc., Otsu, Shiga, Japan). The RNA was reversely-transcribed into cDNA for miRNA detection using miRNA First Strand cDNA Synthesis (Tailing Reaction) kit (B532451-0020, Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China). The reverse transcription quantitative polymerase chain reaction (RT-qPCR) was conducted for synthetized cDNA using Fast SYBR Green PCR kit (Applied Biosystems, Carlsbad, CA) and ABI7500 qPCR instrument (ABI Company, Oyster Bay, NY). GAPDH and U6 were regarded as the internal reference to quantify relative expression using the 2^−ΔΔCt method (Additional file 1: Table S1).

According to the manufacturer's instructions, miRNA was isolated from EVs, tissues, and cells by using the mirVanaTM PARISTM RNA kit (AM1556, Invitrogen, CA, USA). For mRNA analysis, cDNA was synthesized randomly from 1 μg of total RNA using the First Strand cDNA Synthesis Kit (K1622, Fermentas, USA) was used to synthesize cDNA randomly from 1 μg of total RNA; for miRNA analysis, the TaqManTM MicroRNA Reverse Transcription Kit (4366597, Applied Biosystems, USA) was used to synthesize cDNA for miRNA. Finally, the Fast SYBR Green PCR kit (Applied biosystems) and the ABI PRISM 7300 RT-PCR system (Applied biosystems) were used to analyze RNA quantitatively, and each sample was repeated for three replicate wells. miR-30c was used with U6 as the internal reference, and the remaining genes were used with GAPDH. The relative gene expression was analyzed by the 2-ΔΔCt method, calculated as △Ct=CT (target gene)−CT (internal reference) and △△Ct=△Ct (Experimental group)−△Ct (control group), repeated the experiment 3 times, and took the average value. All primers were purchased from Sangon Biotech (Shanghai) Co., Ltd. and the primer sequences were as follows Table 1.

Isolation of total RNA from the cells was performed using Trizol (15596026, Invitrogen) and was reversely transcribed into complementary DNA (cDNA) using PrimeScript RT reagent kit (RR047A) or NCode miRNA First‐Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc). The synthesized cDNA was used to perform RT‐qPCR reactions using Fast SYBR Green PCR Kit (Applied Biosystems) on ABI PRISM 7300 RT‐PCR System (Applied Biosystems). The U6 gene was used as an endogenous control gene for normalizing the expression of miR‐224, whereas glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was taken as the internal reference of other genes. The fold changes between the experiment group and the control group were calculated by means of relative quantification (2^−ΔΔCt method). Primer sequences are shown in Table 1.14, 15

The TRIzol reagents (Invitrogen, Carlsbad, CA, USA) were utilized in order to get the total RNA. cDNA was produced from the isolated RNA by employing the PrimeScript RT Reagent Kit (Takara, Japan) in the process of reverse transcription. Next, synthesized cDNA was subjected to RT-qPCR using the Fast SYBR Green PCR Kit (Applied Biosystems, Foster City, CA, USA) on an ABI PRISM 7300 instrument (Applied Biosystems). 2^−ΔΔCt method was utilized in order to calculate the fold changes in target genes. The primer sequences were presented as follows: CCDC137 5′-ACGGGGCCTATATCCACCG-3′ (forward) and 5′-CGTCGGACTTTATCTAGTCGCC-3′ (reverse); GAPDH 5′-GGAGCGAGATCCCTCCAAAAT-3′ and 5′-GGCTGTTGTCATACTTCTCATGG-3′.

Total RNA was extracted using Trizol reagent (15596026, Invitrogen) and RT was performed using the NCode^TM miRNA First-Strand complementary DNA (cDNA) Synthesis Kit (Thermo Fisher Scientific Inc., Waltham, MA, United States). The synthesized cDNA was subjected to reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection using a Fast SYBR Green PCR Kit (Applied Biosystems, Carlsbad, CA, United States) in an ABI PRISM 7300 RT-qPCR System instrument (Applied Biosystems). Each reaction was conducted in triplicate. The relative expression of miR-497 (normalized to U6) and MUC1 [normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] was calculated using the 2^–ΔΔCT method. Primers are shown in Table 1.